Combination therapies for b-cell lymphomas comprising administration of anti-cd20 antibody

ABSTRACT

New combined therapeutic regimens for treatment of B-cell lymphomas are disclosed which comprise, in particular, administration of anti-CD20 antibodies to patients having low-, intermediate- or high-grade non-Hodgkin&#39;s lymphomas.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/524,837, filed Jun. 15, 2012, which is a divisionalapplication of U.S. patent application Ser. No. 11/840,956, filed Aug.18, 2007 (now U.S. Pat. No. 8,329,172 issued Dec. 11, 2012) which is acontinuation of U.S. patent application Ser. No. 10/196,732, filed Jul.17, 2002 (abandoned), which is a continuation of U.S. patent applicationSer. No. 09/372,202, filed Aug. 11, 1999, (now U.S. Pat. No. 6,455,043issued Sep. 24, 2002) which claims priority under 35 U.S.C. Section119(e) and the benefit of U.S. Provisional Application Ser. No.60/096,180 filed Aug. 11, 1998, the disclosures of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to the use of anti-CD20 antibodies or fragmentsthereof in the treatment of B-cell lymphomas, particularly the use ofsuch antibodies and fragments in combined therapeutic regimens.

BACKGROUND OF THE INVENTION

The use of antibodies to the CD20 antigen as diagnostic and/ortherapeutic agents for B-cell lymphoma has previously been reported.CD20 is a useful marker or target for B-cell lymphomas as this antigenis expressed at very high densities on the surface of malignant B-cells,i.e., B-cells wherein unabated proliferation can lead to B-celllymphomas.

CD20 or Bp35 is a B-lymphocyte-restricted differentiation antigen thatis expressed during early pre-B-cell development and remains untilplasma cell differentiation. It is believed by some that the CD20molecule may regulate a step in the B-cell activation process which isrequired for cell cycle initiation and differentiation. Moreover, asnoted, CD20 is usually expressed at very high levels on neoplastic(“tumor”) B-cells. The CD20 antigen is appealing for targeted therapy,because it does not shed, modulate, or internalize.

Previous reported therapies involving anti-CD20 antibodies have involvedthe administration of a therapeutic anti-CD20 antibody either alone orin conjunction with a second radiolabeled anti-CD20 antibody, or achemotherapeutic agent.

In fact, the Food and Drug Administration has approved the therapeuticuse of one such anti-CD20 antibody, RITUXAN®, for use in relapsed andpreviously treated low-grade non-Hodgkin's lymphoma (NHL). Also, the useof RITUXAN® in combination with a radiolabeled murine anti-CD20 antibodyhas been suggested for the treatment of B-cell lymphoma.

However, while anti-CD20 antibodies and, in particular, RITUXAN® (U.S.;in Britain, MABTHERA®; in general Rituximab), have been reported to beeffective for treatment of B-cell lymphomas, such as non-Hodgkin'slymphoma, the treated patients are often subject to disease relapse.Therefore, it would be beneficial if more effective treatment regimenscould be developed. More specifically, it would be advantageous ifanti-CD20 antibodies had a beneficial effect in combination with otherlymphoma treatments, and if new combined therapeutic regimens could bedeveloped to lessen the likelihood or frequency of relapse. Also, itwould be helpful if current treatment protocols for B-cell lymphoma wereimproved whereby patients with lymphomas which are refractory to othertreatment methods could be treated with chimeric or radiolabeledanti-CD20 antibodies. It would also be helpful if treatment withanti-CD20 antibodies, particularly in combination with other treatments,could be used as therapy for other types of lymphoma besides low grade,follicular non-Hodgkin's lymphoma (NHL).

SUMMARY OF THE INVENTION

The present invention discloses combined therapeutic treatments forB-cell lymphomas, and reports the benefits of treating relapsed orrefractory B-cell lymphomas with chimeric and radiolabeled anti-CD20antibodies. In particular, it has been found that treatment withanti-CD20 antibody provides a beneficial synergistic effect whenadministered in combination with cytokines, radiotherapy, myeloablativetherapy, or chemotherapy. Surprisingly, patients who had prior bonemarrow or stem cell transplantation had an unexpected increase in theover-all response rate when compared with patients with no priortherapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Time to progression (TTP) for all 151 assessable patients andTTP for 76 responders (CR or PR). Kaplan-Meier projected overall medianTTP is 9.0 months (95% confidence interval [CI], 6.7 to 11.4); projectedTTP for responders is 12.5 months (95% CI, 11.0 to 16.0).

FIG. 2. Adverse events attributed to antibody, or cause unknown,stratified by infusion number. As depicted by solid and white shading,96% of events were grade 1 or 2.

DETAILED DESCRIPTION OF THE INVENTION

This invention encompasses combined therapeutic regimens for thetreatment of B-cell lymphomas. In general, such methods include a methodfor treating relapsed B-cell lymphoma, where a patient having priortreatment for lymphoma has relapsed and is administered atherapeutically effective amount of a chimeric anti-CD20 antibody. Suchprior treatments can include, for example, previous treatment withanti-CD20 antibodies, treatments which included a bone marrow or stemcell transplantation, radiotherapy and chemotherapy. The previouschemotherapy may be selected from a wide group of chemotherapeuticagents and combination regimens, including CHOP, ICE, Mitozantrone,Cytarabine, DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La Lachemotherapy regime, ABVD, CEOP, 2-CdA, FLAG & IDA with or withoutsubsequent G-CSF treatment), VAD, M & P, C-Weekly, ABCM, MOPP and DHAP.

Also included in the methods of the invention are methods for treating asubject having B-cell lymphoma wherein the subject is refractory forother therapeutic treatments, including all those listed above, i.e.,treatment with chimeric anti-CD20 antibody, treatments which included abone marrow or stem cell transplantation, radiotherapy and chemotherapy.In particular, encompassed are methods of treating a patient who has notexhibited appreciable tumor remission or regression after administrationof a chimeric anti-CD20 antibody, comprising administering to saidpatient a radiolabeled anti-CD20 antibody.

In particular, the methods of treating a patient with a radiolabeledantibody after a chimeric antibody are performed whereby theradiolabeled anti-CD20 antibody is administered from about one week toabout two years after said administration of said chimeric anti-CD20antibody. More particularly, the radiolabeled anti-CD20 antibody isadministered from about one week to about nine months after saidadministration of said chimeric anti-CD20 antibody.

While any anti-CD20 antibodies can be used for the methods of thepresent invention, a preferred chimeric antibody is C2B8 (IDECPharmaceuticals, Rituximab). A preferred radiolabeled antibody is Y2B8,which is a murine antibody labeled with yttrium-90 (⁹⁰Y). However,antibodies with other radiolabels may be used, particularly thoselabeled with a beta or alpha isotope. Anti-CD 19 antibodies may also beused.

One of skill in the art would know the parameters for choosing aparticular type of anti-CD20 antibody. For instance, chimeric andhumanized antibodies are beneficial for decreased immunogenicity, andfor facilitating antibody effector mediated immune reactions via thehuman constant region domains. Murine and other mammalian antibodies, incontrast, are beneficial for delivering a radiolabel to the tumor cell,as such antibodies generally have a decreased half-life in vivo.

Antibody treatments performed initially to which patients are refractoryor have relapsed may include initial treatments with chimeric antibodiesor mammalian antibodies. Also encompassed are initial treatments withother antibodies, including anti-CD19 antibodies and anti-Lymantibodies, and treatments with antibodies labeled with cytotoxicmoieties, such as toxins, and radiolabels, e.g., ONCOLYM® (Techniclone)or BEXXAR® (Coulter).

It should be clear that the combined therapeutic regimens of the presentinvention can be performed whereby said therapies are givensimultaneously, i.e., the anti-CD20 antibody is administeredconcurrently or within the same time frame (i.e., the therapies aregoing on concurrently, but the agents are not administered precisely atthe same time). The anti-CD20 antibodies of the present invention mayalso be administered prior to or subsequent to the other therapies.Sequential administration may be performed regardless of whether thepatient responds to the first therapy to decrease the possibility ofremission or relapse.

The combined therapies of the present invention include a method fortreating B-cell lymphoma comprising administering at least one chimericanti-CD20 antibody and at least one cytokine. In particular, theinvention includes a method for treating B-cell lymphoma comprisingadministering a synergistic therapeutic combination comprising at leastone anti-CD20 antibody and at least one cytokine, wherein thetherapeutic effect is better than the additive effects of either therapyadministered alone. Preferred cytokines are selected from the groupconsisting of alpha interferon, gamma interferon, IL-2, GM-CSF andG-CSF. Again, the anti-CD20 antibody and the cytokine(s) may beadministered sequentially, in either order, or in combination.

Also included in the present invention is a method for treating B-celllymphoma comprising administering to a patient a therapeuticallyeffective amount of a chimeric anti-CD20 antibody before, during orsubsequent to a chemotherapeutic regimen. Such a chemotherapy regimenmay be selected from the group consisting of, at the very least, CHOP,ICE, Mitozantrone, Cytarabine, DVP, ATRA, Idarubicin, hoelzerchemotherapy regime, La La chemotherapy regime, ABVD, CEOP, 2-CdA, FLAG& IDA with or without subsequent G-CSF treatment), VAD, M & P, C-Weekly,ABCM, MOPP and DHAP.

Also encompassed are methods for treating B-cell lymphoma comprisingadministering to a patient a therapeutically effective amount of achimeric anti-CD20 antibody before, during or subsequent to a bonemarrow or peripheral stem cell transplant. Such bone marrow transplantmay also be accompanied by other therapeutic regimens such aschemotherapy. The antibodies of the present invention may also be usedin a method of reducing residual CD20+ tumor cells in bone marrow orstem cells before or after myeloablative therapy by administering to apatient a chimeric anti-CD20 antibody. It may also be possible to usesuch antibodies in vitro to induce apoptosis of tumor cells and reduceor cure bone marrow or stem cell preparations of residual tumor cellsbefore they are infused back into the patient.

It should be understood that stem cell transplants may be allogeneic orautologous. If the transplant is allogeneic, i.e., from another person,the disclosed therapeutic regimens may include treatments withimmunosuppressive drugs before administration of the anti-CD20antibodies. Coadministration of other drugs designed to enhanceacceptance of the transplant and stimulate the production anddifferentiation of immune cells is also contemplated. For instance, ithas been shown that administration of GM-CSF to marrow transplantrecipients promotes the development of specific bone marrow cells whichin turn produces circulating infection-fighting neutrophils, andincreased the survival rate of marrow transplant recipients.

The methods of the present invention may be used to treat a variety ofB-cell lymphomas, including low grade/follicular non-Hodgkin's lymphoma(NHL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL,intermediate grade diffuse NHL, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL, bulky diseaseNHL and Waldenstrom's Macroglobulinemia. It should be clear to those ofskill in the art that these lymphomas will often have different namesdue to changing systems of classification, and that patients havinglymphomas classified under different names may also benefit from thecombined therapeutic regimens of the present invention.

For instance, a recent classification system proposed by European andAmerican pathologists is called the Revised European American Lymphoma(REAL) Classification. This classification system recognizes Mantle celllymphoma and Marginal cell lymphoma among other peripheral B-cellneoplasms, and separates some classifications into grades based oncytology, i.e., small cell, mixed small and large, large cell. It willbe understood that all such classified lymphomas may benefit from thecombined therapies of the present invention.

The U.S. National Cancer Institute (NCI) has in turn divided some of theREAL classes into more clinically useful “indolent” or “aggressive”lymphoma designations. Indolent lymphomas include follicular celllymphomas, separated into cytology “grades,” diffuse small lymphocyticlymphoma/chronic lymphocytic leukemia (CLL),lymphoplasmacytoid/Waldenstrom's Macroglobulinemia, Marginal zonelymphoma and Hairy cell leukemia. Aggressive lymphomas include diffusemixed and large cell lymphoma, Burkitt's lymphoma/diffuse smallnon-cleaved cell lymphoma, Lymphoblastic lymphoma, Mantle cell lymphomaand AIDS-related lymphoma. These lymphomas may also benefit from thecombined therapeutic regimens of the present invention.

Non-Hodgkin's lymphoma has also been classified on the basis of “grade”based on other disease characteristics including low-grade,intermediate-grade and high-grade lymphomas. Low-grade lymphoma usuallypresents as a nodal disease, and is often indolent or slow-growing.Intermediate- and high-grade disease usually presents as a much moreaggressive disease with large extranodal bulky tumors. Intermediate- andhigh-grade disease, as well as low grade NHL, may benefit from thecombined therapeutic regimens of the present invention.

The Ann Arbor classification system is also commonly used for patientswith NHL. In this system, stages I, II, III, and IV of adult NHL can beclassified into A and B categories depending on whether the patient haswell-defined generalized symptoms (B) or not (A). The B designation isgiven to patients with the following symptoms: unexplained loss of morethan 10% body weight in the 6 months prior to diagnosis, unexplainedfever with temperatures above 38° C. and drenching night sweats.

Occasionally, specialized staging systems are used:Stage I—involvement of a single lymph node region or localizedinvolvement of a single extralymphatic organ or site.Stage II—involvement of two or more lymph node regions on the same sideof the diaphragm or localized involvement of a single associatedextralymphatic organ or site and its regional lymph nodes with orwithout other lymph node regions on the same side of the diaphragm.Stage III—involvement of lymph node regions on both sides of thediaphragm, possibly accompanying localized involvement of anextralymphatic organ or site, involvement of the spleen, or both.Stage IV—disseminated (multifocal) involvement of 1 or moreextralymphatic sites with or without associated lymph node involvementor isolated extralymphatic organ involvement with distant (non-regional)nodal involvement.For further details, see The International Non-Hodgkin's LymphomaPrognostic Factors Project: A predictive model for aggressivenon-Hodgkin's lymphoma. New England J. Med. 329(14): 987-994 (1993).

Preferred antibodies, dosage regimens and particular combinations oftherapy will now be illustrated by way of the following exemplary data.

Rituximab and Y2B8

Non-Hodgkin's lymphoma (NHL) affects approximately 250,000 people in theUnited States. The majority of patients with NHL are not cured bychemotherapy, radiotherapy, or high-dose treatment with autologous bonemarrow (ABMT) or peripheral blood stem cell (PBSC) support.

Approximately 80% of non-Hodgkin's lymphomas are B-cell malignanciesand >95% of these express the CD20 antigen on the cell surface. Thisantigen is an attractive target for immunotherapy because it is foundexclusively on B-cells, and not on hematopoietic stem cells,pro-B-cells, normal plasma cells, or other normal tissues. It is notshed from the cell surface and does not modulate upon antibody binding(1).

Rituximab is one of a new generation of monoclonal antibodies developedto overcome limitations encountered with murine antibodies, includingshort half-life, limited ability to stimulate human effector functions,and immunogenicity (2,3).

Rituximab is a genetically engineered monoclonal antibody with murinelight-and heavy-chain variable regions and human gamma I heavy-chain andkappa light-chain constant regions. The chimeric antibody is composed oftwo heavy chains of 451 amino acids and two light chains of 213 aminoacids and has an approximate molecular weight of 145 kD. Rituximab ismore effective than its murine parent in fixing complement and mediatingADCC, and it mediates CDC in the presence of human complement (4). Theantibody inhibits cell growth in the B-cell lines FL-18, Ramos, andRaji, sensitizes chemoresistant human lymphoma cell lines to diphtheriatoxin, ricin, CDDP, doxorubicin, and etoposide, and induces apoptosis inthe DHL-4 human B-cell lymphoma line in a dose-dependent manner (5). Inhumans, the half-life of the antibody is approximately 60 hours afterthe first infusion and increases with each dose to 174 hours after thefourth infusion. The immunogenicity of the antibody is low; of 355patients in seven clinical studies, only three (<1%) had a detectableanti-chimeric antibody (HACA) response.

Rituximab was genetically engineered using the murine 2B8 antibody. The2B8 antibody has also been conjugated to different radiolabels fordiagnostic and therapeutic purposes. To this end, copending applicationSer. No. 08/475,813 (now U.S. Pat. No. 6,682,734); Ser. No. 08/475,815(now U.S. Pat. No. 6,399,061) and Ser. No. 08/478,967 (now U.S. Pat. No.5,843,439), all herein incorporated by reference in their entirety,disclose radiolabeled anti-CD20 conjugates for diagnostic “imaging” ofB-cell lymphoma tumors before administration of therapeutic antibody.“In2B8” conjugate comprises a murine monoclonal antibody, 2B8, specificto human CD20 antigen, that is attached to Indium[111] (¹¹¹In) via abifunctional chelator, i.e., MX-DTPA (diethylene-triaminepentaaceticacid), which comprises a 1:1 mixture of1-isothiocyanatobenzyl-3-methyl-DTPA and1-methyl-3-isothiocyanatobenzyl-DTPA. Indium-[111] is selected as adiagnostic radionuclide because it emits gamma radiation and finds priorusage as an imaging agent.

Patents relating to chelators and chelator conjugates are known in theart. For instance, U.S. Pat. No. 4,831,175 of Gansow is directed topolysubstituted diethylenetriaminepentaacetic acid chelates and proteinconjugates containing the same, and methods for their preparation. U.S.Pat. Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 of Gansow alsorelate to polysubstituted DTPA chelates. These patents are incorporatedherein in their entirety.

The specific bifunctional chelator used to facilitate chelation inapplication Ser. Nos. 08/475,813, 08/475,815 and 08/478,967 (now U.S.Pat. Nos. 6,682,734; 6,399,061; and. 5,843,439, respectively) wasselected as it possesses high affinity for trivalent metals, andprovides for increased tumor-to-non-tumor ratios, decreased bone uptake,and greater in vivo retention of radionuclide at target sites, i.e.,B-cell lymphoma tumor sites. However, other bifunctional chelators areknown in the art and may also be beneficial in tumor therapy.

Also disclosed in application Ser. Nos. 08/475,813, 08/475,815 and08/478,967 are radiolabeled therapeutic antibodies for the targeting anddestruction of B-cell lymphomas and tumor cells. In particular, the Y2B8conjugate comprises the same anti-human CD20 murine monoclonal antibody,2B8, attached to yttrium-[90] (⁹⁰Y) via the same bifunctional chelator.This radionuclide was selected for therapy for several reasons. The 64hour half-life of ⁹⁰Y is long enough to allow antibody accumulation bythe tumor and, unlike e.g. ¹³¹I, it is a pure beta emitter of highenergy with no accompanying gamma irradiation in its decay, with a rangeof 100 to 1000 cell diameters. The minimal amount of penetratingradiation allows for outpatient administration of ⁹⁰Y-labeledantibodies. Furthermore, internalization of labeled antibodies is notrequired for cell killing, and the local emission of ionizing radiationshould be lethal for adjacent tumor cells lacking the target antigen.

Because the ⁹⁰Y radionuclide was attached to the 2B8 antibody using thesame bifunctional chelator molecule MX-DTPA, the Y2B8 conjugatepossesses the same advantages discussed above, e.g., increased retentionof radionuclide at a target site (tumor). However, unlike ¹¹¹In, itcannot be used for imaging purposes due to the lack of gamma radiationassociated therewith. Thus, a diagnostic “imaging” radionuclide, such as¹¹¹In, can be used for determining the location and relative size of atumor prior to and/or following administration of therapeutic chimericor ⁹⁰Y-labeled antibodies in the combined regimens of the invention.Additionally, indium-labeled antibody enables dosimetric assessment tobe made.

Depending on the intended use of the antibody, i.e., as a diagnostic ortherapeutic reagent, other radiolabels are known in the art and havebeen used for similar purposes. For instance, radionuclides which havebeen used in clinical diagnosis include ¹³¹I, ¹²⁵I, ¹²³I, ⁹⁹Tc, ⁶⁷Ga, aswell as ¹¹¹In. Antibodies have also been labeled with a variety ofradionuclides for potential use in targeted immunotherapy (Peirersz etal. (1987) The use of monoclonal antibody conjugates for the diagnosisand treatment of cancer. Immunol. Cell Biol. 65: 111-125). Theseradionuclides include ¹⁸⁸Re and ¹⁸⁶Re as well as ⁹⁰Y, and to a lesserextent ¹⁹⁹Au and ⁶⁷Cu. I-(131) has also been used for therapeuticpurposes. U.S. Pat. No. 5,460,785 provides a listing of suchradioisotopes and is herein incorporated by reference.

As reported in copending application Ser. Nos. 08/475,813, 08/475,815and 08/478,967 (now U.S. Pat. Nos. 6,682,734; 6,399,061; and. 5,843,439,respectively), administration of the radiolabeled Y2B8 conjugate, aswell as unlabeled chimeric anti-CD20 antibody, resulted in significanttumor reduction in mice harboring a B-cell lymphoblastic tumor.Moreover, human clinical trials reported therein showed significantB-cell depletion in lymphoma patients infused with chimeric anti-CD20antibody. In fact, chimeric 2B8 has recently been heralded the nation'sfirst FDA-approved anti-cancer monoclonal antibody under the name ofRITUXAN®. Thus, at least one chimeric anti-CD20 antibody has been shownto demonstrate therapeutic efficacy in the treatment of B-cell lymphoma.

In addition, U.S. application Ser. No. 08/475,813 (now U.S. Pat. No.6,682,734) herein incorporated by reference, discloses sequentialadministration of RITUXAN®, a chimeric anti-CD20, with both or eitherindium-labeled or yttrium-labeled marine monoclonal antibody. Althoughthe radiolabeled antibodies used in these combined therapies are murineantibodies, initial treatment with chimeric anti-CD20 sufficientlydepletes the B-cell population such that the HAMA response is decreased,thereby facilitating a combined therapeutic and diagnostic regimen.

Thus, in this context of combined immunotherapy, murine antibodies mayfind particular utility as diagnostic reagents. Moreover, it was shownin U.S. application Ser. No. 08/475,813 (now U.S. Pat. No. 6,399,061)that a therapeutically effective dosage of the yttrium-labeled anti-CD20antibody following administration of RITUXAN® is sufficient to (a) clearany remaining peripheral blood B-cells not cleared by the chimericanti-CD20 antibody; (b) begin B-cell depletion from lymph nodes; or (c)begin B-cell depletion from other tissues.

Thus, conjugation of radiolabels to cancer therapeutic antibodiesprovides a valuable clinical tool which may be used to assess thepotential therapeutic efficacy of such antibodies, create diagnosticreagents to monitor the progress of treatment, and devise additionaltherapeutic reagents which may be used to enhance the initial tumorkilling potential of the chimeric antibody. Given the proven efficacy ofan anti-CD20 antibody in the treatment of non-Hodgkin's lymphoma, andthe known sensitivity of lymphocytes to radioactivity, it would behighly advantageous for such chimeric and radiolabeled therapeuticantibodies to find use in combined therapeutic regimens which decreasethe frequency of relapsed or refractory non-Hodgkin's lymphoma. Inaddition, it would be beneficial if such combined therapeutic regimensfound use in the treatment of other B-cell lymphomas.

Low-Grade or Follicular NHL

Single-Agent Studies with Relapsed or Refractory NHL

FDA approval of Rituximab was based on five single-agent studiesprimarily in patients with low-grade or follicular NHL. An early Phase Istudy of single Rituximab infusions ranging from 10-500 mg/m²demonstrated that the maximum tolerated dose had not been reached;however, the length of infusion time at the highest dose was notconsidered feasible for outpatient therapy. The ORR in 15 patients was13% (Table 1)(6).

TABLE 1 Rituximab: Summary of Efficacy Results Median Median Study DRTIP Description Indication N* ORR CR PR (months) (months) ReferencesPhase I/II, Single-Dose Relapsed B-Cell Lymphoma 15 2 (13%) 0 (0%) 2(13%) NA† 8.1  6 Single Agent Phase I/II, Multiple-Dose Relapsed Low-,Intermediate-, 34 17 (50%) 3 (9%) 14 (41%) 8.6 10.2    7 Dose-Rangingand High-Grade Lymphoma Phase II; Multiple-Dose Newly Diagnosed andRelapsed 38 38 (100%) 22 (58%) 16 (42%) 35.3+ 36.7+ 21, 22 Combined withLow-Grade or Follicular B-Cell CHOP Lymphoma Phase III, Multiple-DoseRelapsed Low-Grade or 151 76 (50%) 9 (6%) 67 (44%) 11.6 13.2   8, 9Single-Agent Follicular B-Cell Lymphoma Phase II, Multiple-Dose RelapsedLow-Grade or 35 21 (60%) 5 (14%) 16 (46%) 13.4+ 19.4+ 13 Single-AgentFollicular B-Cell Lymphoma Phase II, Multiple-Dose, Relapsed Low-Gradeor 38 17 (45%) 4 (11%) 13 (34%) 22.3+ 25.2+ 29 Combined with InterferonFollicular B-Cell Lymphoma Phase II, Multiple-Dose, Relapsed Low-Gradeor 28 12 (43%) 1 (4%) 11 (39%) 5.9 8.1 14 Single-Agent Follicular B-CellLymphoma, Bulky Disease Phase II, Multiple-Dose, Relapsed Low-Grade or57 23 (40%) 6 (11%) 17 (29%) 15.0+ 16.7+ 19, 20 Single-Agent FollicularB-Cell Lymphoma, Retreatment Phase II, Multiple-Dose PreviouslyUntreated 30 29 (96%) 19 (63%) 10 (33%) 1 I+ 17+   34 Combined with CHOPIntermediate- or High-Grade Modality Lymphoma Phase II, AlternativeIntermediate- or High-Grade 54 17 (32%) 5 (9%) 12 (22%) NA†  8.2+ 33Multiple Dosing B-Cell Lymphoma

In Phase I of a Phase I/II dose-ranging study, patients received 125-375mg/m² administered as four weekly infusions. No dose-related toxicitieswere demonstrated, and 375 mg/m² was chosen as the Phase II dose. Tumorregressions were observed in 17 of 37 (46%) patients who received thisdose, including 3 (8%) complete responses (CR) and 14 (38%) partialresponses PR (7).

Rituximab Pivotal Trial in Low-Grade or Follicular Lymphoma

Purpose: The CD20 antigen is expressed on more than 90% of B-celllymphomas. It is appealing for targeted therapy, because it does notshed or modulate. A chimeric monoclonal antibody more effectivelymediates host effector functions and is itself less immunogenic than aremurine antibodies.Patients and Methods: This was a multiinstitutional trial of thechimeric anti-CD20 antibody, IDEC-C2B8. Patients with relapsed low gradeor follicular lymphoma received an outpatient treatment course ofIDEC-C2B8 375 mg/m² intravenously weekly for four doses.Results: From 31 centers, 166 patients were entered. Of thisintent-to-treat group, 48% responded. With a median follow-up durationof 11.8 months, the projected median time to progression for respondersis 13.0 months. Serum antibody levels were sustained longer after thefourth infusion than after the first, and were higher in responders andin patients with lower tumor burden. The majority of adverse eventsoccurred during the first infusion and were grade 1 or 2; fever andchills were the most common events. Only 12% of patients had grade 3 and3% grade 4 toxicities. A human antichimeric antibody was detected inonly one patient.Conclusion: The response rate of 48% with IDEC-C2B8 is comparable toresults with single-agent cytotoxic chemotherapy. Toxicity was mild.Attention needs to be paid to the rate of antibody infusion, withtitration according to toxicity. Further investigation of this agent iswarranted, including its use in conjunction with standard chemotherapy.

Patients and Methods Eligibility

Adult patients with relapsed low grade or follicular B-cell lymphoma,histologically confirmed and positive for CD20, were eligible. Patientswith chronic lymphocytic leukemia (lymphocytes>5×10⁹/L) were excluded.Patients had to have either not responded to primary therapy or relapsed(not more than four times), have progressive measurable disease, andsign an institutional review board-approved informed consent. They hadto be at least 3 weeks beyond prior standard therapy includingcorticosteroids, and have recovered from significant toxicities fromprior therapies. Patients had to have good performance status (Zubrod 0to 2) and adequate hematologic, renal, and hepatic function. Patientswere excluded if they had lesions≧10 cm in diameter, CNS lymphoma,AIDS-related lymphoma, pleural effusions or ascites secondary tolymphoma, active opportunistic infection, serious nonmalignant disease,prior investigational therapies including prior anti-CD20 therapy, orrecent major surgery.

Therapy

The antibody dose was 375 mg/m², administered intravenously once weeklyfor a total of four infusions (days 1, 8, 15, and 22) on an outpatientbasis. IDEC-C2B8 was produced and supplied by IDEC Pharmaceuticals Corp.The drug was reconstituted in normal saline to a concentration of 1mg/mL and given through a 0.22s-μm in-line filter. Oral premedicationwith acetaminophen or diphenhydramine was permitted; corticosteroidswere prohibited. The initial infusion rate was 50 mg/h, with subsequentinfusion rate increase if no toxicity was seen. Guidelines werespecified for interruption of infusion, with resumption once adverseevents subsided.

Definition of End Points

Complete response (CR) required the resolution of all symptoms and signsof lymphoma, including bone marrow clearing, for at least 28 days.Partial response (PR) required a ≧50% decrease in the sum of theproducts of perpendicular measurements of lesions, without any evidenceof progressive disease for at least 28 days. Patients who did notachieve a CR or PR were considered nonresponders, even if there was anet decrease (<50%) of measurable disease. Time to progression wasmeasured from the first infusion until progression.

An independent panel of nine radiologists and lymphoma specialistsreviewed and verified all CT scans of patients who exhibited a ≧40%reduction in tumor size as measured by the investigator. This refereedresponse designation is the one used for this report.

Results Response

The overall response rate for the intent-to-treat group of all 166patients was 48%, of which 6% were CRs and the remainder PRs.

A detailed analysis of efficacy was also performed on a subset of 151patients, excluding 15 patients for the following reasons: one neverstarted treatment for personal reasons; eight received one or more dosesof corticosteroids during the evaluation period (a protocol violationthat was strictly enforced to avoid any confusion about the efficacy ofthe antibody); one had surgery within 4 weeks of study entry (anexclusion criterion); one lacked measurable lesions; and four did notcomplete all four doses because of grade 3 or 4 adverse events (theywere included in the toxicity analysis).

The response rate for these 151 assessable patients was virtuallyidentical to that of the intent-to-treat group, with a 50% responserate, including 6% CRs. Among those who did not achieve a CR or PR, themajority (56 of 75) nonetheless had a net decrease of measurable disease(mean decrease, 32%). With a median follow-up duration of 11.8 months,the projected median time to progression for responders is 13.0 monthsfor the intent-to-treat group and 12.5 months for the assessable group(FIG. 1); 53 of 76 responders have not yet relapsed. To date, only ninepatients have died, all of progressive lymphoma.

Table 2 lists response according to clinical features. Significantlylower response rates were noted for patients with the following: SLlymphoma compared with other cell types; positive bone marrow; ≧twoextranodal sites; and, among the subset of 118 patients with follicularlymphomas, those without detectable bcl-2 gene rearrangement by PCR inthe peripheral blood or bone marrow. Unexpectedly, the 23 patients whoseprior therapy had included high-dose regimens with stem-cell or bonemarrow transplantation had a significantly higher response rate thanthose who had not received transplant regimens (78% v 43%, P<0.01).Patients who had achieved a CR or PR with their last prior chemotherapycourse had a nonsignificant but somewhat better response to the antibodythan those who were resistant to chemotherapy (53% v 36%, P=0.06).

TABLE 2 Patient Features and Response No. of % Feature Patients CR + PRP All patients 166 48 — Assessable patients* 151 50 — Age ≧60 years 6751 NS Sex: male 95 48 NS Histology Small lymphocytic 30 13 <.01†Follicular small cleaved 60 60 Follicular mixed 48 60 Follicular largecell 10 60 Other‡ 3 33 Elevated LDH§ 46 43 NS Elevated β₂-microglobulin§ 41 56 NS Bulk§ <5 cm 88 56 NS ≧5 cm 61 43 Marrow§Negative 66 61 .03 Positive 83 42 bcl-2 in peripheral blood¶ Negative 6252 .04 Positive 55 71 bcl-2 in bone marrow¶ Negative 60 52 .05 Positive52 71 ≧2 extranodal site 75 39 .01 Abbreviations: LDH, lactatedehydrogenase; NS, not significant. *Subset analyses conducted on theassessable group of 151 patients (see text); results for theintent-to-treat group were virtually identical. †Comparison of SL versusfollicular histologies. ‡One each with: mucosa-associated lymphoidtissue (MALT); low-grade B-cell lymphoma, not otherwise specified; andmarginal-zone lymphoma. §Data available for LDH on 143, β₂-microglobulinon 148, bulk on 149, and marrow status on 149. ¶For follicular lymphomaonly.

Adverse Events

Adverse events generally occurred during therapy or within the first 30days following therapy (Table 3). The majority were observed during thefirst infusion (FIG. 2) and were grade 1 or 2. After the first infusion,most patients (55%) had no toxicity for the remainder of treatment.

Adverse events were typically brief. The median duration of nausea was 1hour, fever 3 hours, bronchospasm less than 30 minutes, hypotension 1.6hours, and rash and pruritus 2 hours. The antibody infusion rate wastitrated according to adverse events. The mean duration of the firstdose was 5.2 hours (range, 2.5 to 20); 33% of patients hadinterruptions. During the second, third, and fourth doses, the frequencyof interruptions decreased to 6%, 2%, and 1%, respectively, and the meandurations of the infusions were 3.5, 3.3, and 3.3 hours, respectively.

TABLE 3 Adverse Events During Therapy NCI Grade % of Event 1-2 3 4Patients Any 599 18  2 84 General Fever 84 — — 43 Chills 51 2 — 28Headache 26 1 — 14 Asthenia 25 — — 13 Pain 22 — — 11 Pruritus 21 1 — 13Rash 16 — — 10 Urticaria 9 1 — 6 Angioedema 27 1 — 14 Dizziness 11 — — 6Digestive Nausea 34 1 — 18 Vomiting 13 1 — 8 Diarrhea 10 — — 4Respiratory Bronchospasm 15 1 — 8 Dyspnea 1 1 — 1 Rhinitis 14 1 — 7Cough increase 4 1 — 3 Cardiovascular Hypotension 18 1 — 10 Arrhythmia 52 1 2 Hematologic Anemia 1 1 — 1 Thrombocytopenia 5 1 — 3 Leukopenia 121 — 7 Neutropenia 6 — 1 4 NOTE. Includes all grade 3 or 4 events thatwere considered related to the antibody, and most frequent (≧10occurrences) other events, for all patients (N = 165 since 1 patientwithdrew before receiving any antibody). “During Therapy” includes fromday 1 to 30 days after the fourth infusion; for later events, see text.Abbreviation: NCI, National Cancer Institute.

Thirteen patients had hemoglobin levels decrease to as low as 8 to 10g/dL, and four had levels of 7.6 to 7.9; recovery occurred in a medianof 7 days. Three patients with pretreatment platelet counts of 76,000 to85,000×10⁹/L had counts decrease to 63,000 to 72,000 at a median of 19days, with recovery by a median of 7 days; one patient with apretreatment platelet count of 90,000 had a count of 27,000 at day 23,with recovery to 86,000 in 6 days. Fourteen patients had granulocytesdecrease to a level of 1 to 1.5×10⁹/L at median of 41 days, withrecovery by a median of 8 days; two patients had granulocytes decreaseto 0.5 to 0.9 at day 9 and day 23, with recovery to greater than 2.0 by6 to 7 days; one had a granulocyte count of 0.1 at day 51, with recoveryby 4 days. The remainder of patients, 86% of the population, had nocytopenias. Thus, the median (±SE) values for hemoglobin, platelets,leukocytes, and granulocytes remained within normal limits throughoutthe treatment period.

Infections that occurred either during therapy or for up to a yearthereafter were predominantly bacterial (37 of 68), and the vastmajority were minor (61 of 68 grade 1 or 2; none grade 4). Therespiratory tract was the source in 19 and the urinary tract in three;gastroenteritis occurred in three. There were three episodes ofbacteremia, one with Listeria detected before the third infusion, onestaphylococcal, and one polymicrobial, which was felt to becatheter-related; all resolved with antibiotics. Viral infectionsincluded herpes simplex in 10 and herpes zoster in five.

After therapy and during the first year of follow-up evaluation, a totalof 98 related adverse events were reported in 45 patients, of which 81%were grade 1 or 2. The most common late (from 31 days to 1 year afterthe last antibody infusion) adverse events were hematologic: 13neutropenia (five grade 3, one grade 4), 10 leukopenia (one grade 3, nograde 4), and one RBC aplasia.

Discussion

The response rate was 50% with this outpatient four-dose course oftherapy with IDEC-C2B8 for patients with relapsed low grade orfollicular lymphoma.

The toxicity of the current program was notably mild, particularly withrespect to myelosuppressive toxicities that are typical of standardchemotherapy or RIT. Adverse events occurred mainly with the firstinfusion, in a constellation that typically included modest (grade 1 or2) and brief (minutes to hours) fever, chills, and aches. By the secondand subsequent infusions, the majority of patients experienced nofurther infusion-related toxicities.

The overall response rate (ORR) was 48% with a 6% CR and a 42% PR rate(8). The median time to progression (TTP) for responders was 13.2 monthsand the median duration of response (DR) was 11.6 months. Twenty-two of80 (28%) responders remain in ongoing remission at 20.9+ to 32.9+ months(9).

Administration of Rituximab resulted in a rapid and sustained depletionof B-cells. Circulating B-cells were depleted within the first threedoses with sustained depletion for up to six to nine monthspost-treatment in 83% of patients. Median B-cell levels returned tonormal by 12 months following treatment. Although median NK cell countsremained unchanged, a positive correlation was observed between higherabsolute NK cell counts at baseline and response to Rituximab (10).

Several baseline prognostic factors were analyzed to determine theircorrelation to response. Significantly, in 23 patients relapsed afterABMT or PBSC, the ORR was 78% versus 43% in patients who did not undergoprior high-dose therapy (p<0.01). In a multivariate analysis, the ORRwas higher in patients with follicular NHL as compared with smalllymphocytic lymphoma (58% vs. 12%, p<0.01), and higher in patients withchemosensitive relapse as compared with chemoresistant relapse (53% vs.36%, p=0.06). No effect on response rate was associated with: age >60years, extranodal disease, prior anthracycline therapy, or bone marrowinvolvement.

A statistically significant correlation was found between the medianserum antibody concentration and response at multiple time points duringtreatment and follow up (11).

Serum levels of antibody were higher in patients with follicular NHLcompared with small lymphocytic lymphoma. Mean serum antibody was alsoinversely correlated with measurements of tumor bulk and with the numberof circulating B-cells at baseline. The association of lower serumantibody concentrations with higher numbers of circulating NHL cells andwith higher tumor bulk suggest that the main mode of antibody clearanceis to tumor cells. The association of high serum antibody concentrationswith response and lower tumor bulk or circulating cells suggests thathigher or more doses of Rituximab may be necessary to induce responsesin some subsets of patients, such as those with bulky disease.

Nevertheless, responses were seen with Rituximab in 43% of patients withtumors >5 cm and in 35% of patients with tumors >7 cm, suggesting thattreatment of patients with bulky disease with Rituximab is feasible.This is surprising considering it was long thought that antibody therapyis not conducive to treating bulky disease due to the compact nature ofthe tumors.

In a study conducted in Japan (12), patients with relapsed B-celllymphoma were treated with either 250 mg/m² (N=4) or 375 mg/m² (N=8) ofRituximab weekly times four. Of 11 evaluable patients, 8 had follicularNHL, 2 had diffuse large-cell NHL, and one had mantle-cell lymphoma. Twoof the 11 had a CR and 5 had a PR for an ORR of 64%; all responders hadfollicular histology.

Because Rituximab serum levels and response were positively correlatedin previous studies, a Phase II study of eight weekly doses of 375 mg/m²Rituximab was conducted in low-grade or follicular NHL patients. The ORRwas 60% in evaluable patients, with a 14% CR and a 46% PR rate. Medianvalues for TTP in responders and DR were 13.4+ months and 19.4+ months,respectively (13). Though it is difficult to compare across studies, itappears that TTP and DR may be improved by using more doses.

Contrary to early assumptions about antibody therapy being useful onlyin micrometastatic disease, Rituximab® is quite active in high bulkdisease. In a separate study, 31 patients with relapsed or refractory,bulky low-grade NHL (single lesion of >10 cm in diameter) received 375mg/m² Rituximab as four weekly infusions. Twelve of 28 evaluablepatients (43%) demonstrated a CR (1, 4%) or PR (11, 39%)(14).

Waldenstrom's Macroglobulinemia

Waldenstrom's Macroglobulinemia (WM) is a malignancy wherein Blymphocytes secrete excessive amounts of IgM antibodies. WM usuallyoccurs in people over sixty, but has been detected in adults in theirearly thirties. WM today is considered a rare incurable indolentmalignancy, which has in the past been treated by plasmaphoresis toreduce serum viscosity. Chemotherapeutic drugs such as an alkylatingagent and a corticosteroid are often prescribed. The most recommendeddrug for WM has been Leustatin (2CdA).

A report on seven patients with Waldenstrom's macroglobulinemia wherethe patients were treated with Rituximab (375 mg/m² weekly times 4) (15)noted responses in 4 (57%) of patients. Median progression-free survivalwas 8 months (range 3-27+ months). Thus, Rituximab should be useful incombined therapeutic protocols, particularly with chemotherapeuticreagents such as 2CdA.

Chronic Lymphocytic Leukemia (CLL)

CLL is the liquid (leukemic) equivalent of small lymphocytic lymphoma(SLL). Patients with SLL had lower serum levels and a lower responserate when treated with the standard dose of Rituximab than patients withother low-grade NHL subtypes. This is probably due to the very highlevels of circulating tumor cells in patients with CLL, and becausemalignant cells involved in CLL are thought to have reduced levels ofexpression of CD20 on the cell surface.

Nevertheless, the present inventors have discovered that hematologicmalignancies such as CLL may be treated with Rituximab. A recentclinical study evaluated treatment of CLL patients at higher doses ofRituximab (16). All patients receive a first dose of 375 mg/m³ tominimize infusion-relapsed side effects. Subsequent weekly dosages (3)remained the same but were given at an increased dose level. Sixteenpatients have been treated at dosages of 500-1500 mg/m³. Medium age was66 years (range, 25-78). Eighty-one percent had end-stage III-IVdisease. Medium white blood cell count was 40×10⁹/L (range, 4-200), Hgb11.6 g/dl (range, 7.7-14.7), platelets 75×10⁹/L, (range, 16-160), medianβ₂ immunoglobulin was 4.5 mg/L (range, 3.1-9.2). Median numbers of priortherapies was 2.5 (range 1-9). Sixty percent of patients were refractoryto treatment. Two patients developed severe hypertension with the firstdose (375 mg/m²); another one received further therapy. Toxicity atsubsequent escalated dosages has been mild although no patient at the1500 mg/m² dose level has been fully evaluated. Eight patients havecompleted therapy (4 at 500 mg/m², 3 at 650 mg/m², 1 at 825 mg/m²). Onepatient treated at 560 mg/m² achieved full remission. One patient hasprogressive lympocytosis on treatment and all other patients hadreduction in peripheral blood lymphocytosis but less effect on lymphnodes. Dose escalation studies are ongoing.

Another approach to improving response in CLL patients is to upregulatethe CD20 antigen using cytokines. In an in vitro study, mononuclearcells from CLL patients were incubated for 24 hours with variouscytokines. Flow cytometry results showed significant up-regulation byIL-4, GM-CSF, and TNF-alpha (17). In fact, recent data suggests that theupregulation of CD20 observed on CLL cells may be limited to tumor cells(Venogopal et al. Poster—PanPacific Lymphoma meeting, June 1999.Cytokine-induced upregulation of CD20 antigen expression in chroniclymphocytic leukemia (CLL) cells may be limited to tumor cells).Preliminary data also suggest that interferon alpha also upregulatesCD20 on CLL cells after only 24 hours when applied at a concentration of500 to 1000 U/ml.

Thus, by administering certain cytokines to CLL patients prior to orconcurrently with administration of Rituximab, the expression of CD20 onthe surface of malignant B-cells may be upregulated, thereby renderingCD20, as well as other cell surface markers such as CD19, a moreattractive target for immunotherapy. A collaborative study has beeninitiated to test for optimal cytokine doses for CD20 upregulation invivo. The study protocol involves treating ten patients initially withGM-CSF at 250 mcg/m² SQ QD X 3, ten patients with IL-4 mcg/kg SQ QD X 3,and ten patients with G-CSF at 5 mcg/kg SQ QD X 3. Mononuclear cellswill be separated by Ficon Hypaque centrifugation for apoptotic studiesto determine if upregulation of CD20 translates to enhanced killing oftumor cells by Rituximab.

Antibody treatment of CLL can be combined with other conventionalchemotherapeutic treatments known to be useful for the treatment of CLL.The most frequently used single agent for CLL is chlorambucil(leukeran), given either as 0.1 mg/kg daily or 0.4 to 1.0 mg/kg every 4weeks. Chlorambucil is often combined with oral prednisone (30 to 100mg/m²/d), which is useful in the management of autoimmune cytopenias.Cyclophosphamide is an alternative to chlorambucil, the usual dose being1-2 g/m² every 3-4 weeks together with vincristine and steroids (e.g.,COP regimen).

Various drug combinations have been used for CLL, including COP(cyclophosphamide, Oncovin, and prednisone), and CHOP (these three drugsplus doxorubicin). Fludarabine has shown an effect in the treatment ofCLL, and gave an ORR of 50% in a group of patients treated with 25-30mg/m²/d every 3-4 weeks. http://www.cancernetwork.com. Although somepatients have been shown to be refractory for fludarabine. Such patientsmay also be resistant to 2-CdA because often, patients who arerefractory to fludarabine are also refractory to 2-CDA (O'Brien et al.N. Engl. J. Med. 330: 319-322 (1994)).

Hence, anti-CD20 antibody therapy will be particularly useful forpatients who are refractory or who have relapsed after treatment withchemotherapeutic drugs. Rituximab therapy may also be combined withradiotherapy in these patients. TBI with a low fraction size of 15 cGyto total doses of 75 to 150 cGy has been shown to be effective in aboutone-third of patients.

A Phase II trial is currently being conducted by CALGB in CLL patients.Rituximab and fludarabine are administered concurrently, followed byRituximab consolidation versus fludarabine induction followed byRituximab.

Rituximab with Myeloablative Therapy

Myeloablative therapy has yielded responses in indolent lymphomas;however, residual tumor cells may remain despite high-dose therapy andthe PBSC reinfused may contain tumor cells. Rituximab is being usedbefore stem cell mobilization and after transplant to reduce residualCD20+ tumor cells and contamination of the bone marrow or stem cellsharvested. Interim results demonstrated that no CD20+ cells weredetectable in harvested cells. Eighteen of 24 patients achievedengraftment and the treatment was well tolerated. PCR testing is ongoingto evaluate residual tumor cells (18).

Retreatment of Relapsed Low-Grade NHL with Rituximab

A trial evaluating retreatment of 53 patients who had responded toRituximab and later relapsed has been reported (19). Seven of 56evaluable patients (13%) obtained a CR and 16 a PR (29%), for an ORR of42%. Four patients who had a second response received a third treatment;3 of these responded.

After treatment with two courses of Rituximab, one patient's tumor,initially classified as follicular, small cleaved cell NHL, no longerexpressed the CD20 antigen and was unresponsive to Rituximab at the timeof transformation to diffuse, large-cell NHL (20).

Thus, while retreatment with Rituximab is effective for treatingpatients who have relapsed after prior treatment with Rituximab, theremay be an increased incidence of CD20-tumor cells after secondarytreatment. This observation supports the utility of the combinedtherapeutic treatment regimens described herein.

Combination of Rituximab and CHOP Chemotherapy for Low-Grade NHL

Chemotherapy with cyclophosphamide, doxorubicin, vincristine, andprednisone (CHOP) is an effective first-line therapy for low-grade orfollicular NHL. Though initial response rates are high, relapseeventually occurs and subsequent chemotherapy regimens produceremissions with shorter durations. A Phase II trial was initiated toevaluate the combination of CHOP and Rituximab (21) in newly diagnosedand relapsed low-grade or follicular NHL because their mechanisms ofaction are not cross-resistant, and Rituximab is synergistic withcertain cytotoxic drugs, including doxorubicin (5).

Twenty-nine of 38 patients received no prior anticancer therapy. CHOPwas administered at standard doses every three weeks for six cycles withsix infusions of Rituximab (375 mg/m²). Rituximab infusions 1 and 2 wereadministered on Days 1 and 6 before the first CHOP cycle, which startedon Day 8. Rituximab infusions 3 and 4 were given 2 days before the thirdand fifth CHOP cycles, respectively, and infusions 5 and 6 were given onDays 134 and 141, respectively, after the sixth CHOP cycle.

In this combination study, 100% of the 38 patients treated responded(CR, 58%; PR, 42%). Of 35 evaluable patients who completed treatment,there were 63% CR, and 37% PR(21). Median DR is 35.3+ months with medianprogression-free survival not reached after a median observation time of36.7+ months. Twenty patients are still in remission after 36+ months to53.4+ months (22). This DR is impressive even for first-line treatment,and 24% of this trial population had relapsed after chemotherapy.

In a study to be conducted by CALGB, 40 patients with low-grade NHL willreceive Rituximab weekly times 8 and oral cyclophosphamide dailystarting on Day 8. Twenty patients will receive Rituximab alone for 8weekly doses.

A Phase III study conducted by ECOG in patients with low-grade NHL iscomparing the combination of cyclophosphamide and fludarabine (Arm A)with standard CVP therapy (Arm B). In the randomization to Arm A or ArmB, patients are stratified by age, tumor burden, histology, and Bsymptoms. Responders in both arms will undergo a second randomization toRituximab maintenance therapy (375 mg/m² weekly times 4 every 6 monthsfor 2 years (Arm C) or to observation (Arm D).

Combination of Rituximab with Cytokines

Rituximab Plus Interferon Alpha

Interferon is a cytokine involved in modulating the immune system (23).Mechanisms by which interferon may increase the effectiveness ofantibodies include the potentiation of antigen expression (24),increased targeting of antibodies into tumors (25,26), and enhancedcytotoxicity of immunotoxins (27).

In a combination trial, interferon-alpha (Roferon-A), a cytokine with asingle-agent clinical activity in NHL (28), and Rituximab were given topatients with relapsed low-grade or follicular NHL. Interferon-alpha(2.5 or 5 MIU) was administered subcutaneously, three times weekly for12 weeks. Rituximab® was administered by IV infusion weekly for fourdoses (375 mg/m²) starting on the fifth week of treatment. The ORR was45% (17/38 patients); 11% had a CR and 34% had a PR. Kaplan-Meierestimates of the median DR and TTP in responders were 22.3+ and 25.2+months, respectively (29). Previous combination studies ofinterferon-alpha and chemotherapeutic regimens containing anthracyclinesyielded prolonged time to progression, but did not consistently increaseresponse or survival rates (30-32). These early results suggest that thecombination of Rituximab and interferon-alpha may prolong the time toprogression relative to Rituximab alone.

Rituximab Plus G-CSF

In a separate study, Rituximab and G-CSF are being evaluated in relapsedlow-grade NHL. It has been demonstrated in vitro as well as in vivo inhealthy volunteers that G-CSF, via its effect on myeloid precursorcells, induces FcRI-positive neutrophils that are capable of functioningas effector cells in ADCC. Therefor, a Phase VII study was initiated toevaluate the toxicity and efficacy of the combined treatment.

Both in Phase I and Phase II, patients were administered a standard doseof G-CSF (5 μg/kg/day) administered for three days, starting 2 daysbefore administration of Rituximab. Phase I consisted of a doseescalation of Rituximab (125, 250, or 375 mg/m² weekly X4). Earlyresults in 9 patients evaluated so far yielded an ORR of 67% (44% CR,22% PR) with minor toxicity in 8 of the 9 patients (33). The mostfrequent adverse events were fever (4/8 patients), rhinitis (4/8),chills (3/8) and headaches (3/8), which were comparable to the adverseevents observed previously in administration of Rituximab alone. ThePhase II part of the study has been initiated, which will examine theefficacy of the combination of G-CSF and 375 mg/m² Rituximab X4.

Rituximab Plus IL-2

High-dose therapy with autologous peripheral blood stem cells (PBSC) orbone marrow (BM) rescue has been used to treat NHL, however successremains limited by the high risk of relapse, which is 50-80%. In aneffort to improve durable remissions post-transplant, immunotherapyincluding high dose and low dose therapy with IL-2 has been studied in anumber of treatment centers. Such studies have suggested that IL-2therapy does demonstrate early post-transplant anti-Tumor activity.

Initially following autologous transplant, patients display delayedimmune reconstitution which potentially results in diminishedimmune-mediated tumor eradication (43, 44). Indeed, it has been shownthat both CD$+T cells and cytotoxic CD8+ T cells are depressed (45-49).In vitro assays have demonstrated a profound suppression of T cellcytolytic and proliferative responses as well as decreased production ofIL-2 in response to mitogens and soluble antigens. However, soluble IL-2is able to restore these immune responses suggesting that immune cellsin patients after autologous transplant are capable of responding toexogenous IL-2 (47). Peripheral blood NK activity also remains lowerfollowing BMT than control values and the NK activity is also augmentedby addition of exogenous IL-2 (49). These data suggest thatadministration of IL-2 to patients shortly after stem cell transplantmay enhance immune responsiveness at a critical period when tumor burdenis minimal and when immune responsiveness in the absence of IL-2 islacking.

For instance, Caligiuru et al. have shown that IL-2 (Hoffman-LaRoche)administered at 0.45×10⁶ U/M²/day by 24 hour CIV for 12 weeks was ableto expand the absolute number of CD56 bright NK cells (50-52). Thisregimen was administered to non-transplant patients in the outpatientsetting with little toxicity.

Animal models have shown that non-LAK inducing low doses of IL-2dramatically enhances anti-tumor activity when administered withtumor-specific T effector cells (53). In addition, Soiffer et al. (54)administered low doses of IL-2 to 13 autologous BMT or T cell depletedallogeneic BMT recipients undergoing treatment for relapsed leukemia orlymphoma. Enhanced immunological responsiveness was demonstrated in thelaboratory with a 5- to 40-fold increase in circulating CD56 brightCD16+ CD3-NK cells. Moreover, this low dose regimen of IL-2 resulted inaugmented in vitro killing of the NK targets K562. When Soiffer et al.(55) updated the outcome of 29 allogeneic BMT patients who received lowdose IL-2, they found superior survival for these patients (70%)compared to histological controls (30%, p=0.41).

Lauria et al. (56) treated 11 patients with high grade NHL at a medianof 42 days after ABMT with IL-2 at a dose of 2×10⁶ IU/m² god for twoweeks and then 3×10⁶ IU/m² twice a week for a year. Phenotypic analysisshowed a persistent and significant (p=0.001) increase in the proportionand absolute number of total lymphocytes and especially of both CD16 andCD56 NK cells after 6 months of therapy. None of the patients progressedwith a median follow-up of twenty-two months (range 10-42 months) afterstarting therapy. In addition, two patients with residual disease afterABMT, one in the liver and second in the lymph nodes, obtained acomplete response after 7 and 10 months of IL-2 therapy.

Vey et al. (57) treated 25 patients with refractory or relapsed HD (11patients) and NHL (14 patients) with low dose IL-2.48% of the patientshad resistant disease at transplant and 84% achieved CR after ABMT. IL-2was started at a mean of 54 days after transplant and consisted of afirst cycle of 5 days followed by 4 cycles of 2 days every other week.Patients received a mean of 160×10⁶ IU/m² of IL-2. After a five yearfollow-up, the probability of survival and DFS is 72% (HD 73% and NHL70%) and 45% (HD 36% and NHL 48%).

A group at the Fred Hutchinson Cancer Research Center (FHCRC) hasrecently found that low dose IL-2 therapy was well-tolerated in theoutpatient setting, and that remissions in patients treated with lowdose IL-2 tended to be longer than without IL-2 treatment. IL-2 therapywas associated with an increase in the number of certain populations ofimmune cells, including CD8+ CD69+ cells; CD16+ CD8+ cells; CD16+ CD69+cells; CD16+ CD56+ cells; CD16+ CD122+ cells; CD16+ Dr+ cells; and CD8+CD56+ cells. There was also an increase in the expression of lyticactivity against the tumor targets K562 and Daudi, with a median of5.9-fold and 6.5-fold increase, respectively. Relapses, when theyoccurred, occurred at a median of 17.8 months after transplant, andtherefor remissions were reported to be characteristically longer thanwhat was historically seen in transplant recipients without IL-2therapy.

Given the encouraging data gathered from single therapy studies withIL-2 on ABMT transplant recipients, it seemed reasonable to combine IL-2therapy with Rituximab post transplant, given that Rituximab'sbiological activity appears to be mediated through ADCC andcomplement-mediated lytic activity. Thus, a Phase I trial has beeninitiated in collaboration with the FHCRC to evaluate the safety andpotential efficacy of a combined therapeutic regimen.

A separate Phase II study is also being performed to evaluate theefficacy and the incidence of HACA formation in patients receivinglow-dose IL-2 and RITUXAN®. A specific objective of this study is toassess whether ADCC is enhanced by in vivo exposure to IL-2 and whetherADCC activity correlates with clinical response. Inclusion criteria forpatients are histologically confirmed stage II-IV low-grade, follicularB-cell or mantle cell lymphoma. Mantle cell lymphoma, for the purposesof this clinical study, is defined as CD5+, CD23-(if available) and/orbcl−1+ by immunohistochemistry. Patients who did not respond to or haverelapsed following their first treatment with a standard therapy, i.e.,chemotherapy, radiotherapy, ABMT and/or immunotherapy, are eligible.

Rituximab Plus GM-CSF for the Treatment of Relapsed Low Grade orFollicular B-Cell Lymphoma

Two separate Phase II trials have also been initiated to test theefficacy of combined treatment with Rituximab and GM-CSF. One studyinvolves 40 patients with relapsed low grade B-cell lymphoma, andcomprises administering Rituximab at 375 mg/m² weekly×4 (d. 1, 8, 15,22) and GM-CSF (Leukine, Immunex) at 250 mcg sc three times weekly for 8weeks, starting one hour before the first dose of Rituximab. This studywill be used to evaluate the clinical efficacy (overall response rate(ORR), overall complete response rate, time to progression andfailure-free survival) of the combined therapeutic regimen, tocharacterize the safety (qualitative, quantitative, duration andreversibility of adverse events) of the combined therapy, and todetermine the effects of the combined therapy on relevant lymphocytesubsets and cytokines. The second study plans to also monitorimmunologic parameters to assess the mechanism of killing (complement C3and C4, CHSO, flow cytometry for CD3, CD4, CD8, CD16, CD19 and CD56 andADCC assay).

Rituximab Plus Gamma-Interferon

Gamma-interferon may also be useful in combined therapy with Rituximabfor treating patients with low-grade or higher-grade lymphomas. It ishas recently been found that gamma-interferon upregulates CD20expression on multiple myeloma (MM) patient plasma cells, patientB-cells, as well as on normal donor B-cells (Treon et al., Lugano,1999). In fact, Treon and colleagues have shown that gamma-interferonaugments binding of these cells to Rituximab. Induction of CD20expression on plasma cells occurred in a dose dependent manner, withupregulation seen with as little as 1 U/ml of interferon gamma A plateauoccurred at 100 U/ml at 48 hours. Thus, gamma-interferon may also bebeneficial when administered in combination with Rituximab.

Intermediate-Grade and High-Grade NHL Single-Agent Studies

In a study conducted in Europe and Australia, alternative dosingschedules were evaluated in 54 relapsed or refractory intermediate- orhigh-grade NHL patients (34). Rituximab was infused at 375 mg/m² weeklyfor 8 doses or at 375 mg/m² once followed by 500 mg/m² weekly for 7doses. The ORR was 31%; (CR 9%, PR 22%) no significant differencebetween the dosing regimens was observed. Patients with diffuselarge-cell lymphoma (N=30) had an ORR of 37% and those with mantle-celllymphoma (N=12) had an ORR of 33%.

Combination of Rituximab and CHOP Chemotherapy

In another study, 31 patients with intermediate- or high-grade NHL (19females, 12 males, median age 49) received Rituximab on Day 1 of each ofsix 21-day cycles of CHOP (35). Of 30 evaluable patients, there were 19CR (63%) and 10 PR (33%), for an ORR of 96%. This regimen was consideredwell tolerated and may result in higher response rates than withRituximab or CHOP alone.

The NCI Division of Cancer Treatment and Diagnosis is collaborating withIDEC Pharmaceuticals Corporation to explore Rituximab treatment in otherindications. A Phase II trial of CHOP versus CHOP and Rituximab is beingconducted by ECOG, CALGB, and SWOG in older patients (>60 years) withmixed, diffuse large cell, and immunoblastic large cell histology NHL(N=630 planned). This study includes a secondary randomization tomaintenance with Rituximab versus non-maintenance.

A Phase III trial of Rituximab and CHOP in 40 patients with previouslyuntreated mantle-cell lymphoma is also ongoing at the Dana FarberInstitute. Rituximab® is administered on Day 1 and CHOP is given on Days1-3 every 21 days for 6 cycles. Accrual for this study has beencompleted. A Phase II trial of CHOP followed by Rituximab in newlydiagnosed follicular lymphoma conducted by SWOG has also been completed.Results of these two trials are being analyzed.

A Phase II trial of CHOP and Rituximab versus CHOP alone in HIV-relatedNHL conducted by the AIDS Malignancy Consortium is ongoing; 120 patientsare planned.

Rituximab after Myeloablative Therapy Relapse

Rituximab has shown promising early results in patients with relapsedintermediate-grade NHL after high-dose therapy with autologous PBSCsupport. Six of seven patients responded (1 CR and 5 PR) and one patienthad stable disease; therapy was well tolerated (36).

Safety Experience

Adverse events and clinical laboratory data from 315 patients in thefive single-agent U.S. studies were combined to provide a safety profileof Rituximab in patients with low-grade or follicular NHL. The majorityof adverse events were infusion-related and occurred with decreasingfrequency after the first infusion. The most common infusion-relatedevents were fever (49%), chills (32%), nausea (18%), fatigue (16%),headache (14%), angioedema (13%), pruritus (10%), and occasionally,hypotension (10%) and bronchospasm (8%). During the treatment period (upto 30 days following the last dose), 10% of patients experienced Grade 3or 4 adverse events, which were primarily infusion-related orhematologic. Thrombocytopenia (<50,000 platelets/mm³) occurred in 1.3%of patients, neutropenia (<1000/mm³) occurred in 1.9%, and anemia (<8gm/dL) occurred in 1.0%. Although Rituximab induced B-cell depletion in70%-80% of patients, abnormally decreased serum immunoglobulins wereobserved in a minority of patients and the incidence of infection didnot appear to be increased.

Hypotension requiring interruption of the Rituximab infusion occurred in10% of patients and was Grade 3 or 4 in 1%. Angioedema was reported in13% of patients and was considered serious in one patient. Bronchospasmoccurred in 8% of patients; 2% were treated with bronchodilators. Asingle report of bronchiolitis obliterans was noted. Most patientsexperienced no further infusion-related toxicities by the second andsubsequent infusions. The percentage of patients reporting adverseevents upon retreatment was similar to that reported following the firstcourse (14).

Four patients developed arrhythmias during Rituximab infusion. One ofthe four discontinued treatment because of ventricular tachycardia andsupraventricular tachycardias. The other three patients experiencedtrigeminy (N=1) and irregular pulse (N=2) and did not requirediscontinuation of therapy. Angina was reported during infusion andmyocardial infarction occurred four days post-infusion in one subjectwith a prior history of myocardial infarction.

The overall incidence of adverse events and Grade 3 and 4 adverse eventswas higher in patients with bulky disease than in patients withnon-bulky disease. The incidence of dizziness, neutropenia,thrombocytopenia, myalgia, anemia, and chest pain was higher in patientswith lesions >10 cm. The incidence of Grade 3 or 4 neutropenia, anemia,hypotension, and dyspnea was also higher in patients with bulky diseasecompared with patients with lesions <10 cm (19).

Since FDA approval of Rituximab for treatment of relapsed or refractorylow-grade or follicular NHL in 1997, an estimated 17,000 patients havebeen treated. In May, 1998, descriptions of eight post-marketing reportsof severe infusion-related adverse events associated with the use ofRituximab that resulted in fatal outcomes were summarized. In seven ofthe eight fatalities, severe symptoms occurred during the firstRituximab infusion. The cause of death was not reported or remainsunknown for two of the eight cases. Severe respiratory events, includinghypoxia, pulmonary infiltrates, or adult respiratory distress syndromecontributed to six of the eight reported deaths. One patient had apretreatment lymphocyte count of 600,000/mm³; another, a creatinine of8; a third, a respiratory rate of 40; and a fourth, pancytopenia.Patients with a high tumor burden or with a high number of circulatingmalignant cells may be at higher risk and these patients should bemonitored closely throughout each infusion.

Most of the adverse events recently described were previously observedin Rituximab clinical studies. One notable exception is aninfusion-related syndrome associated with rapid tumor lysis, that wasreported in six patients with high numbers of circulating tumor cells(37,38). This syndrome was characterized by fever, rigors, bronchospasmwith associated hypoxemia, a rapid decline in peripheral lymphocytes,laboratory evidence of tumor destruction, and transient, severethrombocytopenia. These patients had diagnoses of B-prolymphocyticleukemia (N=2), chronic lymphocytic leukemia (N=2), mantle-cell lymphoma(N=1), or transformed NHL (N=1); all had elevated circulatinglymphocytes, bulky adenopathy, and organomegaly. Although five of thesesix patients required hospitalization, symptoms resolved and subsequentRituximab treatments were well tolerated; the last patient refusedfurther therapy and died of progressive disease two weeks later.

In a separate report of seven patients with CLL and one patient withmantle-cell lymphoma, tumor lysis syndrome was observed after the firstRituximab infusion in those patients with lymphocyte counts >10×10⁹ L,(39).

Radioimmunotherapy with ⁹⁰Yttrium-Labeled Anti-CD20 Antibody inCombination with Rituximab

Another therapeutic approach to NHL under evaluation is a radiolabeledanti-CD20 antibody (IDEC-Y2B8) in combination with Rituximab. IDEC-Y2B8(⁹⁰Y-ibritumomab tiuxetan) is a murine IgG₁ kappa anti-CD20 antibodyconjugated to ⁹⁰Y via a chelator, MX-DTPA, which is covalently bound tothe antibody. Rituximab (250 mg/m2) is administered prior to IDEC-Y2B8to deplete peripheral B lymphocytes and improve biodistribution of theradiolabeled antibody.

In a recently reported Phase I/II study (40-42), patients with low-gradeNHL (N=34), intermediate-grade NHL (N=14), or mantle-cell lymphoma (N=3)were treated with IDEC-Y2B8. The median age was 60, 71% were male, and96% were Caucasian. Of 51 patients with relapsed or refractory NHL, 34(67%) responded to single doses of 0.2, 0.3, or 0.4 mCi/kg of IDEC-Y2B8.The ORR was 82% (28/34) for patients with low-grade or follicular NHLand was 43% (6/14) for patients with intermediate-grade lymphoma. Nopatients with mantle-cell disease responded.

A Phase III randomized study comparing IDEC-Y2B8 with Rituximab (375mg/m² weekly times 4) for treatment of low-grade follicular ortransformed NHL patients is ongoing. Another Phase III trial is alsobeing conducted in patients with relapsed NHL who are refractory toRituximab.

SUMMARY

In the absence of curative therapy for NHL, the objective of treatmentis to achieve control of the disease for a meaningful duration andprovide relief of tumor-related symptoms without undue toxicity.Treatment with Rituximab is a brief, 22-day outpatient therapy withlimited adverse events in most patients. In clinical studies, 50% ofevaluable relapsed or chemotherapy refractory low-grade or follicularNHL patients achieved complete or partial responses. These responseswere durable without maintenance therapy; the median TTP for responderswas 13.2 months and the median DR was 11.6 months in the pivotal study.

Rituximab is approved as a safe and effective treatment for patientswith relapsed low-grade or follicular B-cell NHL. It has significantclinical activity, a novel mechanism of action, and compares favorablywith alternative therapies in response rate and response duration.Completion of ongoing studies will verify the role of alternativeRituximab regimens and Rituximab in the treatment of other CD20+B-lymphocyte malignancies.

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What is claimed is:
 1. A method of extending median time to progressionfor responders by at least 13 months in patients with relapsed orrefractory, low grade or follicular, CD20-positive, B-cell non-Hodgkin'slymphoma, comprising administering four doses of 375 mg/m² of rituximabto the patients.
 2. A method according to claim 1, wherein the patientsexhibit an overall response rate to the administration of about 48%.